EP0855447B1 - Verfahren zur Bestimmung von Nukleinsäuresequenzen - Google Patents

Verfahren zur Bestimmung von Nukleinsäuresequenzen Download PDF

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Publication number
EP0855447B1
EP0855447B1 EP98300481A EP98300481A EP0855447B1 EP 0855447 B1 EP0855447 B1 EP 0855447B1 EP 98300481 A EP98300481 A EP 98300481A EP 98300481 A EP98300481 A EP 98300481A EP 0855447 B1 EP0855447 B1 EP 0855447B1
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Prior art keywords
nucleic acid
rna
dna
specific nucleic
probe
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EP98300481A
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English (en)
French (fr)
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EP0855447A2 (de
EP0855447A3 (de
Inventor
Takahiko Ishiguro
Juichi Saitoh
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Tosoh Corp
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Tosoh Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Definitions

  • the present invention relates to a method of detection or quantification of a specific nucleic acid anticipated to be in a gene mixture containing DNA or RNA, and is useful for gene diagnosis in the field of clinical diagnosis, for cloning useful genes and for exploring unknown genes.
  • the present invention is also useful as a method of optimizing the reaction conditions for the amplification of genes.
  • Assays of a specific nucleic acid using a probe which is complementary in base sequence to the nucleic acid utilize the ability of the specific nucleic acid to hybridize with the probe.
  • the sandwich assay uses two probes which hybridize with different parts of the specific nucleic acid.
  • one of the probes is immobilized on a solid support, while the other is labeled with a dye which is visible by its color or a fluorescent substance or an enzyme which catalyzes production of such a dye or fluorescent substance.
  • These probes are added to a sample and allowed to hybridize with the specific nucleic acid in the sample so that a complex of the three is formed on the solid support. Then, the solid support is separated from the supernatant of the sample solution to separate the unhybridized second probe (B/F separation step).
  • the label in the complex on the solid support is measured to detect and quantify the specific nucleic acid in the sample.
  • an enzyme which catalyzes production of a dye which is visible by its color or a fluorescent substance is used to label the second probe, after the formation of the complex, the unhybridized second probe is removed, and a precursor of the dye or the fluorescent substance which is a substrate for the enzyme, is added to the sample solution. The dye or the fluorescent substance produced as the reaction product is measured to detect and quantify the nucleic acid in the sample.
  • the sandwich assay uses a solid support in the reaction mixture
  • the second probe can become non-specifically adsorbed on the solid support.
  • the presence of the label of the second probe non-specifically adsorbed on the solid support produces errors in the measurement of the labeled hybrid on the solid support, which causes problems in detection and quantification of a specific nucleic acid.
  • diagnosis of virus infections requires sensitive and reproducible detection of trace amounts of virus nucleic acids in clinical samples, the above-mentioned problem attributable. to non-specific adsorption is a serious problem to be solved.
  • the product of the amplification by PCR is a double-stranded DNA. Therefore, in order to hybridize the probes and the amplified nucleic acid, a heating procedure for melting the double-stranded DNA amplification product into single strands after addition of the probes to the PCR reaction mixture (denaturing) and a subsequent cooling procedure for formation of a double-stranded DNA from the probe DNA and the target DNA (annealing) are essential. Therefore, additional labor and more analysis time are necessary for practical clinical diagnoses, which usually pursue effectiveness and economies.
  • an assay method which does not use such a support, does not involve denaturing and annealing procedures at the time of measuring the label of the probe, and minimizes carryover of the nucleic acid amplified by PCR.
  • a support-free assay method which comprises conducting PCR in the presence of a fluorescent intercalative dye and measuring the fluorescence intensity of the reaction solution is known (Japanese Unexamined Patent Publication JP-A-5-237000, Igaku-no Ayumi 173(12), 959-963(1995), and Analytical Biochemistry, 229, 207-213(1995)). Because the PCR products are double-stranded DNA, a fluorescent intercalative dye which alters the fluorescent property (for example increases the fluorescent intensity) on intercalation with a double-stranded nucleic acid, is added to a sample solution before amplification by PCR and the fluorescence intensity of the reaction solution is monitored to detect or quantify the target nucleic acid before the amplification.
  • This method makes it possible to follow the progress of PCR by measuring the fluorescence intensity of reaction solutions in sealed vessels, and can obviate the problem of false positive results attributable to carryover of the amplification products because it does not require sampling of reaction solutions from reaction vessels.
  • the above-mentioned assay by performing PCR in the presence of a fluorescent intercalative dye and monitoring the fluorescence intensity of a PCR reaction solution is excellent as a support-free, or homogeneous one-step assay.
  • the assay method has a problem that intercalation of a fluorescent intercalative dye with the genomic DNA gives a high background count and thereby makes it difficult to accurately measure the change in the fluorescence intensity attributable to the amplification of the specific nucleic acid.
  • the two nucleic acids which are complementary to specific regions of a specific nucleic acid and used as the primers for elongation in PCR can bind complimentarily, depending on their sequences, and in such a case, serve as a template for each other to produce a primer dimer. Because a fluorescent intercalative dye non-specifically intercalates also to the primer dimer, the increased background attributable to the non-specific intercalation is a problem in monitoring the change in the fluorescent property based on amplification of the specific nucleic acid.
  • a method of assaying nucleic acids has been developed by using a fluorescent intercalative dye-labeled probe capable of recognizing a specific nucleic acid sequence which comprises a single-stranded oligonucleotide complementary in nucleic acid sequence to the specific nucleic acid sequence of the target specific nucleic acid and a fluorescent intercalative dye linked to the single-stranded oligonucleotide as a label.
  • the probe is designed so that, when the single-stranded oligonucleotide hybridizes with the specific nucleic acid, the intercalative dye intercalates with the resulting double-stranded oligonucleotide to alter the fluorescent property.
  • the present inventors have conducted studies to provide a method for support-free or homogeneous assay of a specific nucleic acid by using a fluorescent intercalative dye, which is specific enough for the specific nucleic acid sequence to allow a precise assay even if the sample contains a large amount of genomic DNA or even if a primer dimer is produced during amplification by PCR prior to the assay, and which can detect or quantify the specific nucleic acid sequence at a constant temperature without separation of excess probe and the denaturing and annealing procedures, and as a result, have accomplished the present invention.
  • the present invention provides a method of assay of a specific nucleic acid anticipated in a sample, which comprises:
  • double-stranded DNA consisting of a promoter sequence for an RNA polymerase and a specific nucleic acid sequence following the promoter sequence is produced by using the specific nucleic acid.
  • a pair of primers which are complementary in sequence to the specific nucleic acid sequence are used, and one of them is a promoter primer which has a promoter sequence for an RNA polymerase at the 5'- end.
  • a DNA polymerase and deoxyribonucleoside triphosphates are also used.
  • the above-mentioned double-stranded DNA is produced by DNA elongation reaction by the repeated action of DNA polymerase by using the above-mentioned two primers.
  • the DNA polymerase used in the present invention is not particularly limited, and for example, E. coli DNA polymerase III, the Klenow flagment, T4 DNA polymerase, T7 DNA polymerase, Thermus aquaticus DNA polymerase, Thermus Thermophilus DNA polymerase and the like may be used.
  • the promoter primer used in the present invention is designed to have at least the promoter sequence for an RNA polymerase and a sequence complementary to the specific nucleic acid sequence in this order from the 5' end toward the 3' end, as described above.
  • the segment which is complementary to the specific nucleic acid is complementary to at least a part of the specific nucleic acid, not necessarily to the entire specific nucleic acid sequence.
  • the segment may be from 6 to 100 nucleotides long, preferably from 10 to 30 nucleotides long in order to ensure the specificity for a nucleic acid complementary to the specific nucleic acid.
  • the promoter sequence for an RNA polymerase and the sequence complementary to the specific nucleic acid sequence may be linked via a few bases (linker bases) which are related to neither of them.
  • the above-mentioned DNA elongation reaction is preceded by synthesis of cDNA by using the specific nucleic acid as a template in the DNA producing step.
  • the above-mentioned two primers containing sequences complementary to the specific nucleic acid sequence, a reverse transcriptase and the substrates for the reverse transcriptase, deoxyribonucleoside triphosphates are used.
  • Either of the primers used in the DNA producing step may be used for the synthesis of cDNA, and in this case, the other primer has to be added to the reaction solution for the synthesis of double-stranded DNA by a DNA polymerase subsequent to the synthesis of cDNA.
  • the reverse transcriptase is not particularly limited, and those commercially available can be used. Further, in the case of a DNA polymerase having a reverse transcription activity, it is possible to perform the synthesis of cDNA and the synthesis of double-stranded DNA without discontinuity by adding a reagent containing the above-mentioned two primers, the DNA polymerase and deoxyribonucleoside triphosphates to a sample.
  • RNA producing and measuring step subsequent to the DNA producing step a single-stranded RNA is synthesized by the action of an RNA polymerase on the synthesized double-stranded DNA and measured.
  • This step is initiated in the presence of at least a RNA polymerase, ribonucleoside triphosphates and a fluorescent intercalative dye-labeled probe complimentary to the resulting RNA after the DNA producing step. Therefore, the RNA producing and measuring step can be initiated only by adding these reagents after the DNA producing step.
  • the probe hybridizes with the resulting RNA to alter the fluorescence of the fluorescent intercalative dye. Therefore, it is possible to detect the specific nucleic acid in the sample and determine its initial amount by measuring the fluorescence intensity of the reaction mixture.
  • the RNA polymerase used in the RNA producing and measuring step is not particularly limited, and for example, those commercially available such as T7 RNA polymerase, T3 polymerase and SP6 RNA polymerase may be used.
  • the probe used in the present invention is an oligonucleotide which hybridizes selectively with the RNA synthesized by the action of the RNA polymerase and is labeled with a fluorescent intercalative dye which alters the fluorescence on binding to double-stranded DNA (Japanese Patent Application JP7-185599, EP-A-714986, Nucleic Acid Research, 24(24), 4992-4997(1996)).
  • the fluorescent intercalative dye is not particularly limited, as long as it alters the fluorescence on binding to double-stranded DNA. However, those which enhance the fluorescence on intercalation are preferable in view of the ease of monitoring, and particularly, thiazole orange, oxazole yellow and their derivatives are preferable because they show radical alteration in fluorescence.
  • the fluorescent intercalative dye is linked to the oligonucleotide by a covalent bond, if necessary, via a linker of an appropriate length.
  • a linker of an appropriate length any linkers that do not hinder the fluorescent intercalative dye from binding to double-stranded DNA may be used, difunctional hydrocarbons having functional groups at both ends are preferred because they are easy to link to oligonucleotides.
  • a commercial kit C6-Thiolmodifier, Clontech
  • the fluorescent intercalative dye as the label may be linked to any site of the nucleotide, including the 5'- end, the 3'- end and the center, as long as the linkage neither hinders the fluorescent intercalative dye from intercalating to double-stranded DNA nor hinders the oligonucleotide from hybridizing with the RNA.
  • the region of the probe which is complimentary to the RNA is preferably from 6 to 100 nucleotides, and more preferably from 10 to 30 nucleotides long in order to ensure the specificity for the RNA.
  • the method of the present invention makes it possible to precisely detect and quantify a target nucleic acid in samples by monitoring the fluorescence intensity of the reaction mixture without denaturing and annealing procedures for hybridization.
  • the fluorescent intercalative dye as the label of the probe alters fluorescence on hybridization of the probe with the RNA produced depending on the initial amount of the specific target nucleic acid in the sample, and it is possible to detect and quantify the hybrid without separation of the unhybridized excess probe, and to provide a simple homogeneous one-step assay of a nucleic acid having a specific sequence.
  • the DNA producing step of the present invention may utilize PCR.
  • the double-stranded DNA sequence having a promoter sequence for RNA polymerase upstream and a specific nucleic acid sequence downstream is amplified greatly in the DNA producing step and is then subjected to the RNA producing and measuring step.
  • the ordinary PCR process for denaturing and annealing comprising heating and cooling is conducted by using the above-mentioned two primers in the DNA producing step.
  • Figure 1 illustrates an embodiment of the present invention wherein the specific nucleic acid in the sample is a single-stranded RNA.
  • a reverse transcriptase synthesizes the cDNA of the specific nucleic acid in the presence of a primer for reverse transcription and deoxyribonucleoside triphosphates.
  • the synthesized cDNA is complimentary to the specific nucleic acid sequence.
  • PCR is conducted by adding a promoter primer having the same sequence as the 5'- end of the specific nucleic acid and a thermoresistant DNA polymerase to give a double-stranded DNA having a promoter region for an RNA polymerase at the 5'- end.
  • the RNA producing and measuring step follows.
  • a reagent containing a fluorescent intercalative dye-labeled probe, an RNA polymerase and ribonucleoside triphosphates is added to the reaction solution, and then the reaction solution is incubated at the optimum temperature for the RNA polymerase.
  • the RNA polymerase transcribes the double-stranded DNA having a promoter region produced in the DNA producing step into an RNA having the specific nucleic acid sequence.
  • the RNA produced by transcription hybridizes with the fluorescent intercalative dye-labeled probe coexisting in the reaction solution to enhance the fluorescence intensity in proportion to the amount of the hybrid. Therefore, it is possible to detect the specific nucleic acid or determine the initial amount of the specific nucleic acid by measuring the fluorescence intensity of the reaction solution before and after, or during this step.
  • PCR in the presence of a pair of the primers and a promoter primer, deoxyribonucleoside triphosphates and thermoresistant DNA polymerase to produce a double-stranded DNA having a promoter region for an RNA polymerase is followed by the above-mentioned procedures.
  • the method of the present invention was applied to assay of a recombinant HCV RNA, and its lower limit of detection was evaluated.
  • Figure 2 shows the increases in fluorescence during 30 minutes and the detection ratio.
  • the increase in fluorescence intensity for 10 copies was significantly distinguished from that for 0 copy (t test, significance level 1%) and 10 copies of the recombinant HCV RNA were detectable.
  • Figure 3 shows the correlation between the amounts of DNA calculated from the densities of the ethidium bromide-stained bands obtained by agarose electrophoresis of portions of the reaction mixtures immediately after the addition of SP6 RNA polymerase and the increases in fluorescence intensity. A good correlation was found between the amounts of the PCR products and the increases in fluorescence intensity.
  • Serum samples from 20 patients with chronic hepatitis C were assayed by the method of the present invention, and the results were compared with those obtained by using a commercial HCV RNA detection kit (Amplicore HCV, Japan Rosh).
  • the method of the present invention facilitates detection of specific nucleic acids in clinical samples as compared with the conventional method.
  • the method of the present invention was applied to assay of a recombinant HCV RNA, and its lower limit of detection was evaluated.
  • Figure 4 shows the increases in fluorescence intensity and the detection ratios. All the samples containing at least 10 copies of the recombinant HCV RNA were judged positive. 87.5% of the samples containing 5 copies were judged positive.
  • the present invention allows detection and quantification of specific nucleic acids anticipated to be in samples by a simple homogeneous assay without using supports and separating the unhybridized excess probe in the RNA producing and measuring step which involves monitoring of fluorescent intensity. Further, because the present invention removes the necessity of denaturing and annealing in the step and only requires addition of the necessary reagent to sample solutions and monitoring of fluorescence intensity at a constant temperature in the step, the step can be performed only by one operation, namely, bringing the reagent into contact.
  • the fluorescent intercalative dye as the label of the probe alters fluorescence, (for example enhances the fluorescence intensity) on hybridization of the probe with the RNA (which contains the same base sequence as the specific nucleic acid sequence) produced in the RNA producing and measuring step, it is possible to detect and quantify the resulting hybrid without separating the unhybridized excess probe. Therefore, use of vessels made of a material optically transparent at the excitation and emission wavelength of the fluorescent intercalative dye allows continuous assay while removing the problem of the chance of false positive results due to carryover of the amplification product because there is no need to collect reaction solutions from vessels.
  • the probe since the probe specifically recognizes a specific nucleic acid sequence and alters fluorescence on hybridization, if PCR is performed in the DNA producing step, the probe does not hybridize with a primer dimer produced as a byproduct. Therefore, the primer dimer does not contribute to the change in the fluorescence of the reaction solution. Accordingly, sequence-specific assay of a specific nucleic acid can be achieved without separation of the primer dimer after PCR by monitoring the fluorescence of the reaction solution.
  • the present invention obviates cumbersome operations such as transferring a reaction solution from one vessel to another and provides a quite simple assay method useful for clinical diagnosis.

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Claims (4)

  1. Verfahren zur Bestimmung einer spezifischen Nucleinsäure, die in einer Probe vermutet wird, welches umfasst:
    einen DNA-Herstellungsschritt, der die Herstellung einer doppelsträngigen DNA mit einer Promotor-Sequenz für eine RNA-Polymerase und mit der Nuclein-Sequenz der spezifischen Nucleinsäure (spezifische Nucleinsäuresequenz) stromabwärts von der Promotor-Sequenz unter Verwendung der spezifischen Nucleinsäure in der Probe als Matrize umfasst, wobei der DNA-Herstellungsschritt die DNA-Amplifikation durch Polymerasekettenreaktion (PCR), bei der die doppelsträngige DNA mit der spezifischen Nucleinsäuresequenz in Gegenwart einer DNA-Polymerase und eines zu der spezifischen Nucleinsäuresequenz komplementären Paares von Primern synthetisiert wird, umfasst, wobei einer der Primer ein Promotor-Primer mit einer Promotorsequenz für eine RNA-Polymerase am 5'-Ende ist; und
    einen RNA-Herstellungs- und -Meßschritt, die die Herstellung einer einzelsträngigen RNA mit der spezifischen Nucleinsäuresequenz durch die RNA-Polymerase und die Messung der einzelsträngigen RNA umfassen,
       wobei der RNA-Herstellungs- und -Meßschritt unmittelbar auf den DNA-Herstellungsschritt folgen, und:
    a) durch Zugabe von mindestens der RNA-Polymerase, Ribonucleosidtriphosphaten und einer Sonde, die mit einem fluoreszierenden Interkalationsfarbstoff markiert ist und zu der einzelsträngigen RNA komplementär ist, zu der im DNA-Herstellungsschritt erhaltenen Reaktionslösung gestartet werden,
    b) die Messung der Fluoreszenzintensität der Reaktionslösung umfassen,
    c) bei konstanter Temperatur durchgeführt werden, und
    d) kein Denaturierungs- und Reassoziierungsverfahren zur Hybridisierung oder Abtrennung der nicht mit der hergestellten einzelsträngigen RNA hybridisierten Sonde umfassen.
  2. Verfahren nach Anspruch 1, wobei sich die mit einem fluoreszierenden Interkalationsfarbstoff markierte Sonde bei Hybridisierung mit der RNA, die durch die RNA-Polymerase hergestellt wurde, im Vergleich mit der Fluoreszenzeigenschaft, die die Sonde aufweist, wenn die Sonde nicht mit der RNA hybridisiert wurde, in der Fluoreszenzeigenschaft ändert.
  3. Verfahren nach Anspruch 1 oder 2, wobei die mit einem fluoreszierenden Interkalationsfarbstoff markierte Sonde dadurch gekennzeichnet ist, dass sich der fluoreszierende Interkalationsfarbstoff zwischen Sonde und RNA, die durch die RNA-Polymerase bei Hybridisierung von Sonde und RNA hergestellt wurde, einlagert.
  4. Verfahren nach einem vorhergehenden Anspruch, wobei die spezifische Nucleinsäure eine einzelsträngige RNA ist und im DNA-Herstellungsschritt ein Paar von Primern, einschließlich des Promotor-Primers, verwendet wird, und das die Herstellung einer cDNA, die zu der spezifischen Nucleinsäuresequenz komplementär ist, unter Verwendung einer reversen Transkriptase in Gegenwart von mindestens einem der Primer und von Deoxyribonucleosidtriphosphaten und die Synthese einer doppelsträngigen DNA mit der spezifischen Nucleinsäuresequenz in Gegenwart des anderen Primers und einer DNA-Polymerase umfaßt.
EP98300481A 1997-01-24 1998-01-23 Verfahren zur Bestimmung von Nukleinsäuresequenzen Expired - Lifetime EP0855447B1 (de)

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EP03027996A EP1400598A1 (de) 1997-01-24 1998-01-23 Verfahren zur Bestimmung von Nukleinsäuresequenzen

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JP1099697 1997-01-24
JP10996/97 1997-01-24
JP01099697A JP3968810B2 (ja) 1997-01-24 1997-01-24 核酸配列分析方法

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EP03027996A Division-Into EP1400598A1 (de) 1997-01-24 1998-01-23 Verfahren zur Bestimmung von Nukleinsäuresequenzen

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EP0855447A2 EP0855447A2 (de) 1998-07-29
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EP0855447B1 true EP0855447B1 (de) 2004-03-31

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JP2002153289A (ja) * 2000-11-21 2002-05-28 Tosoh Corp ジェノグループｉｉ型小型球形ウイルスを特徴づける塩基配列および検出のためのオリゴヌクレオチド
JP2003116543A (ja) * 2001-10-10 2003-04-22 Tosoh Corp 薬効と毒性の評価法
KR100459394B1 (ko) * 2001-10-30 2004-12-03 엘지전자 주식회사 인터컬레이터를 이용한 핵산의 전기화학발광 검출방법
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WO2004040019A1 (ja) 2002-10-29 2004-05-13 Riken 核酸の増幅法
EP2415878A1 (de) 2003-12-25 2012-02-08 Riken Verfahren zur Amplifikation von Nukleinsäure und Verfahren zur Erkennung von mutierter Nukleinsäure damit
US20080108059A1 (en) * 2004-07-30 2008-05-08 Tosoh Corporation Method Of Measuring Heterogeneous Nuclear Ribonucleoprotein B1 (Hnrnp B1) Mrna
JP2006223194A (ja) * 2005-02-17 2006-08-31 Tosoh Corp スタニオカルシン１（ＳＴＣ１）ｍＲＮＡの測定方法
CN101137759A (zh) * 2005-02-18 2008-03-05 独立行政法人科学技术振兴机构 基因检测方法
JP2007116999A (ja) * 2005-10-28 2007-05-17 Tosoh Corp ＲｅｇＩＶｍＲＮＡの測定方法
JP5481841B2 (ja) * 2008-11-28 2014-04-23 東ソー株式会社 サイトケラチン１９ｍＲＮＡの測定方法
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JP3968810B2 (ja) 2007-08-29
EP0855447A2 (de) 1998-07-29
EP0855447A3 (de) 1999-04-14
DE69822685T2 (de) 2004-08-19
JPH10201476A (ja) 1998-08-04
US6063572A (en) 2000-05-16
DE69822685D1 (de) 2004-05-06
EP1400598A1 (de) 2004-03-24

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